Biology subjects

Corcoran, W. K.

Publications and source records attributed to Corcoran, W. K..

4 recordsLinked to original sources

bioRxiv2024

Exploring structure-function relationships in engineered receptor performance using computational structure prediction

Engineered receptors play increasingly important roles in transformative cell-based therapies. However, the structural mechanisms that drive differences in performance across receptor designs are often poorly understood. Recent advances in protein structural prediction tools have enabled the modeling of virtually any user-defined protein, but how these tools might build understanding of engineered receptors has yet to be fully explored. In this study, we employed structural modeling tools to perform post hoc analyses to investigate whether predicted structural features might explain observed functional variation. We selected a recently reported library of receptors derived from natural cytokine receptors as a case study, generated structural models, and from these predictions quantified a set of structural features that plausibly impact receptor performance. Encouragingly, for a subset of receptors, structural features explained considerable variation in performance, and trends were largely conserved across structurally diverse receptor sets. This work indicates potential for structure prediction-guided synthetic receptor engineering.

synthetic biology↗

bioRxiv2024

Conversion of natural cytokine receptors into orthogonal synthetic biosensors

Synthetic receptors enable bioengineers to build cell-based therapies that perform therapeutic functions in a targeted or conditional fashion to enhance specificity and efficacy. Although many synthetic receptors exist, it remains challenging to generate new receptors that sense soluble cues and relay that detection through orthogonal mechanisms independent of native pathways. Towards this goal, we investigated co-opting natural cytokine receptor ectodomains into Modular Extracellular Sensor Architecture receptors (yielding natural ectodomain, NatE MESA receptors). We generated multiple high-performing, orthogonal synthetic cytokine receptors, identified design principles and constraints, and propose guidance for extending this approach to other natural receptors. We demonstrate utility of NatE MESA by engineering T cells to sense an immunosuppressive cue and respond with customized transcriptional output to support CAR T-cell activity. Finally, we multiplex NatE MESA to logically evaluate multiple cues associated with the tumor microenvironment. These technologies and learnings will enable engineering cellular functions for new applications.

synthetic biology↗

bioRxiv2020

RNA sequence and structure determinants of Pol III transcriptional termination in human cells

The precise mechanism of transcription termination of the eukaryotic RNA polymerase III (Pol III) has been a subject of considerable debate. Although previous studies have clearly shown that at the end of RNA transcripts, tracts comprised of multiple uracils are required for Pol III termination, whether upstream RNA secondary structure in the nascent transcript is necessary for robust transcriptional termination is still subject to debate. We sought to address this directly through the development of an in cellulo Pol III transcription termination assay using a synthetic biology approach. Specifically, we utilized the recently developed Tornado expression system and a stabilized Corn RNA aptamer to create a Pol III-transcribed RNA that produces a detectable fluorescent signal when transcribed in human cells. To study the effects of RNA sequence and structure on Pol III termination, we systematically varied the sequence context upstream of the aptamer and identified sequence characteristics that enhance or diminish termination. We found that in the absence of predicted secondary structure, only poly-U tracts longer than then the average length found in the human genome (4-5 nucleotides), efficiently terminate Pol III transcription. We found that shorter poly-U tracts could induce termination when placed in proximity to secondary structural elements, while secondary structure by itself was not sufficient to induce termination. These findings demonstrate a key role for sequence and structural elements within Pol III-transcribed nascent RNA for efficient transcription termination, and demonstrate a generalizable assay for characterizing Pol III transcription in human cells.

molecular biology↗

bioRxiv2019

Computation-guided optimization of split protein systems

Splitting bioactive proteins, such as enzymes or fluorescent reporters, into conditionally reconstituting fragments is a powerful strategy for building tools to study and control biochemical systems. However, split proteins often exhibit a high propensity to reconstitute even in the absence of the conditional trigger, which limits their utility. Current approaches for tuning reconstitution propensity are laborious, context-specific, or often ineffective. Here, we report a computational design-driven strategy that is grounded in fundamental protein biophysics and which guides the experimental evaluation of a focused, sparse set of mutants--which vary in the degree of interfacial destabilization while preserving features such as stability and catalytic activity--to identify an optimal functional window. We validate our method by solving two distinct split protein design challenges, generating both broad insights and new technology platforms. This method will streamline the generation and use of split protein systems for diverse applications.

synthetic biology↗